Allergic conditions now affect almost 1 in 3 individuals during their lifetime and pose a major socio-economic burden on society. These conditions, including asthma, allergic rhinitis, eosinophilic esophagitis, atopic dermatitis, and intestinal allergy most often have a genetic basis. Genetic susceptibility alone cannot account for these conditions but gene-environment interactions are responsible for the induction/inception of an allergic disease and for the maintenance and progression of the disease. Understanding the mechanisms underlying these conditions is central to developing appropriate treatment strategies and even preventative interventions. Defining the pathophysiology of these atopic diseases/conditions has, on one hand been very fruitful, with identification of critical cell-cell interactions, mediators such as cytokines and chemokines, and unique signaling pathways, but direct targeting of many of these circuits has not sustained in the clinic. To a large extent this may be the result of targeting individual downstream processes and not their upstream control or convergence points. Attempts to block key mediators singly such as anti-histamines, cytokines (IL-4, IL-5, IL-13) or cells (eosinophils) have had limited success. The primary therapy for treatment of these diseases/conditions remains corticosteroids, a therapy without downstream specificity but multiple actions upstream in the pathways. Corticosteroids are not immunomodulatory but are anti-inflammatory. Moreover, when stopped, all disease manifestations return. New therapeutic approaches are needed, especially those that have the potential to modify existing disease and prevent progression.
The survival kinases are defined as cytoplasmic serine/threonine kinases that phosphorylate substrates which contribute to the control of cell proliferation, survival, differentiation, apoptosis, and tumorigenesis (Amaravadi, R., et al. 2005. J. Clin Invest 115:2618-2624). Akt is a well-studied survival kinase, in which gene amplifications have been demonstrated in several cancers (Cheng, J. Q., et al. 1992. Proc. Natl. Acad. Sci. USA 89:9267-9271; Bellacosa, A. D., et al. 1995. Int J. Cancer 64:280-285; Stahl., J. M., et al. 2004. Cancer Res. 64:7002-7010) and inhibitors assessed for treatment of malignancy (Masure, S. B., et al. 1999. Eur. J. Biochem. 265:353-360; Yang, L., et al. 2004. Cancer Res. 64:4394-4399; Kondapaka, S. B., et al. 2003. Mol. Cancer Ther. 2:1093-1103). The provirus integration site for Moloney murine leukemia virus (PIM), Pim kinase is another potent survival kinase that has been implicated in cell survival through suppression of myc-induced apoptosis (van Lohuizen, M., et al. 1989. Cell 56:673-682). There are three subtypes of Pim kinases (serine/threonine kinases) that control cell survival, proliferation, differentiation, and apoptosis (Bachmann, M., et al. 2005. Int. J. Biochem. Cell Biol. 37:726-730; Wang, Z., et al. 2001. J. Vet. Sci. 2:167-179; Amaravadi R., et al. 2005. J. Clin Invest. 115:2618-2624). Pim1 kinase and Pim2 kinase are primarily restricted to hematopoietic cells and Pim3 kinase primarily is expressed in brain, kidney, and mammary tissue (Mikkers, H., et al. 2004. Mol Cell Biol 24:6104-6115). Unlike other serine/threonine kinases, these kinases are regulated via JAK/STAT activation driven transcription of the Pim gene rather than by membrane recruitment and phosphorylation (Fox, C. J., et al. 2005. J. Exp. Med. 201:259-266). Overexpression of Pim kinase has been demonstrated in various human lymphoma, leukemic and prostatic cancers and the role of Pim-induced oncogenic transformation has been extensively studied in hematopoietic tumors (Amson, R., et al. 1989. Proc. Natl. Acad. Sci. USA 86:8857-8861; Valdman, A., et al. 2004. Prostrate 60:367-371; Cibull, T. L., et al. 2006. J. Clin. Pathol. 59:285-288; Nieborowska-Skorska, M., et al. 2002. Blood 99:4531-4539). Despite intensive studies on the role of Pim kinase in the development of tumor cells, the role of Pim kinase in immune cells has been less well studied. In human, Pim kinases are expressed in eosinophils, and play a major role in IL-5-induced eosinophil survival (Temple, R., et al. 2001. Am. J. Respir. Cell Mol. Biol. 25:425-433; Andina, N., et al. 2009. J. Allergy Clin. Immunol. 123:603-611). In addition, Pim1 expression was increased in eosinophils from BAL fluid compared to blood from asthmatic patients after allergen provocation (Stout, B. A., et al. 2004. J. Immunol 173:6409-6417). In a recent study, Pim kinase was also shown to promote cell survival in T cells (Fox, C. J., et al. 2005. J. Exp Med. 201:259-266).
Pim1 kinase is involved in cell proliferation and differentiation (Wang Z, et al. J Vet Sci. 2001; 2(3):167-179) and has been implicated in cytokine-dependent signaling in hematopoietic cells and T cells (Aho T L, et al. BMC Cell Biol. 2006; 7:21-29; Rainio E M, et al. J Immunol. 2002; 168(4): 1524-7). It has been showed that Pim1 expression is enhanced during T cell activation in a protein kinase C dependent manner (Wingett D, et al. J Immunol. 1996; 156(2):549-57). Pim1 increased T cell proliferation by enhancing activity of nuclear factor activated T-cells (NFAT) thereby increasing IL-2 production in T cells (Rainio E M, et al. J Immunol. 2002; 168(4): 1524-7).
Although it is well known that survival kinases regulate common substrates like Bad or 4EBP1 to induce cell survival and proliferation (Yan, B., et al. 2003. J. Biol. Chem. 278:45358-45367), the downstream activities of each kinases are different. To date, the precise downstream target of Pim kinase is not known. However, c-Myc, suppressor of cytokine signaling-1 (SOCS-1), PAP-1, PTP-U2S, and heterochromatin protein 1 (HP-1) all are potential downstream targets of Pim kinase (van Lohuizen, M., et al. 1989. Cell 56:673-682; Chen, X. P., et al. 2002. Proc. Natl. Acad. Sci. USA 99:2175-2180; Maita, H., et al. 2000. Eur. J. Biochem. 267:5168-5178; Koike, N., et al. 2000. FEBS Lett 467:17-21; Wang, Z., et al. 2001. Arch Biochem Biophys 390:9-18). Recently, nuclear factor of activated T-cells (NFATc1) was reported to be a potential downstream substrate of Pim kinase (Rainio, E. M. et al. 2002. J. Immunol 168:1524-1527). As the regulation of NFAT activity has been shown to be important for normal selection of thymocytes, NFAT may play a role in the functional development of T cells (Patra, A. K. 2006. J. Immunol. 177:4567-4576) as well as in the suppression of CD4+ and CD8+ T cell proliferation and T cell cytokine production as a downstream substrate of Pim kinase.
CD4+ T cells play a central role in controlling allergic inflammation (Buss, W. W., et al. 1995. Am J Respir Crit Care Med. 152:388-393). CD4+ T cells, especially Th2 cells producing IL-4, IL-5, and IL-13, have been identified in BAL fluid and airway tissues in asthmatics (Robinson, D. S., et al. 1992. N. Engl J. Med. 326:298-304). The transfer of Th2 cells followed by airway allergen challenge in mice was sufficient to induce airway eosinophilia and AHR (Cohn, L., et al. 1997. J. Exp. Med. 186:1731-1747; Hogan, S. P., et al. 1998. J. Immunol. 161:1501-1509). Conversely, CD8+ T cells, which are also key components of adaptive immunity, have drawn limited attention in the pathogenesis of asthma. However, recent studies demonstrated the increased numbers of CD8+ T cells in the lung tissues of asthmatics (Azzawi, M., et al. 1990. Am. Rev. Respir. Dis. 142:1407-1413) and recent reports suggested that not only CD4+ T cells but also CD8+ T cells were essential to the development of AHR and allergic inflammation (Hamelmann, E., et al. 1996. J. Exp Med. 183:1719-1729; Isogai, S., et al. 2004. J. Allergy. Clin. Immunol. 114:1345-1352; Miyahara, N., et al. 2004. J. Immunol. 172:2549-2558; Miyahara, N., et al. 2004. Nat. Med. 10:865-869). Subsets of CD8+ T cells, which produce IL-4, IL-5, and IL-13 but not IFN-γ, labeled as Tc2 cells, are known to increase AHR and airway inflammation (Croft, M., et al. 1994. J. Exp Med. 180:1715-1728; Seder, R. A., et al. 1992. J. Immunol. 148:1652-1656; Coyle, A. J., et al. 1995. J. Exp. Med. 181:1229-1233). Thus, both CD4+ T cells and CD8+ T cells play key roles in the pathogenesis of asthma.
Asthma is a multifactorial inflammatory disorder characterized by persistent airway inflammation and airway hyperresponsiveness (AHR) as a result of the cellular and molecular responses induced by allergen exposure, infectious pathogens, or chemical agents (Buss, W. W., et al. 2001. N. Engl. J. Med. 344:350-362; Umetsu, D. T., et al. 2002. Nat. Immunol. 3:715-720). Several clinical and experimental investigations have shown that T cells, especially Th2-type cells, play a pivotal role in the development of AHR and eosinophilic inflammation through the secretion of a variety of Th2 cytokines, including IL-4, IL-5, and IL-13 (Wills-Karp, M., et al. 1998. Science 282:2258-2261; Robinson, D. S., et al. 1992. N. Engl. J. Med. 326:298-304). These cytokines bind to the extracellular Janus kinase (JAK) receptors and subsequently induce the phosphorylation and activation of signal transducers and activators of transcription (STAT), which translocates into the nucleus, where it binds to DNA and affects basic cell functions, cellular growth, differentiation and death Aaronson, D. S., et al. 2002. Science 296:1653-1655.
Knowledge of the pathogenesis of atopic diseases/conditions was originally interpreted within the framework of a binary T helper 1 (Th1)/Th2 paradigm. This has now been broadened to incorporate other T cell subsets. Importantly, the differentiation and commitment of these populations of T cells is shaped by transcriptional circuits that center on key transcriptional regulators, the proteins that bind DNA to activate or repress gene expression. Runt-related transcription factors (Runx), are a novel family of transcription factors which are key regulators of lineage-specific gene expression, and are responsible for the development of allergic responses (Fainaru, O., et al. 2004. EMBO J. 23:969-979; Fainaru, O., et al. 2005. Proc. Natl. Acad. Sci. USA 102:10598-10603). There are three mammalian Runx genes: Runx1, Runx2, and Runx3. Runx1 is required for hematopoiesis (Okuda, T., et al. 1996. Cell 84:321-330) and Runx2 is critical regulator of osteogenesis (Ducy, P., et al. 1997. Cell 89:747-745). The Runx3 gene resides on human chromosome 1p36.1 (Levanon, D., et al. 1994. Genomics 23:425-432), which maps to a region containing susceptibility genes for asthma (Haagerup, A., et al. 2002. Allergy 57:680-686) and on mouse chromosome 4 (Calabi, F., et al. 1995. Genomics 26:607-610), which contains a susceptibility gene for atopic dermatitis (Christensen U., et al. 2009. 126:549-557). Runx3 is thought to play a critical role in regulating T-cell development, the differentiation of Th1/Th2 cells and Th1/Th2 cytokine production and the development of an allergic disease/condition. It has been reported that Pim1 kinase regulates Runx expression in vitro (Aho T L, et al. BMC Cell Biol. 2006; 7:21-29) and loss of Runx3 results in spontaneous development IBD, as well as allergic asthma (Brenner O, et al. Proc Natl Acad Sci USA. 2004; 101(45):16016-21; Fainaru O, et al. EMBO J. 2004; 23(4): 969-79). The Runx transcription factors are also key regulators of lineage-specific gene expression (Komine O, et al. J Exp Med. 2003; 198 (1): 51-61).
Peanut allergy is one of the most common food allergies characterized by acute allergic diarrhea and intestinal inflammation. During an allergic reaction, several cell types, including Th2 cells (T-helper cells), mast cells, and eosinophils, are recruited to the intestine and activated to release cytokines and chemokines, contributing to increased intestinal inflammation (Kweon M N, et al. J Clin Invest 2000; 106:199-206; Wang M, et al. J. Allergy Clin Immunol 2010, 126 (2): 306-316). CD4+T cells (i.e. T cells that express CD4), especially Th2 cells, which are known to produce interleukin-4 (IL-4) and interleukin-13 (IL-13), are considered critical in the development of allergic diarrhea and intestinal inflammation (Knight A K, et al. Am J Physiol Gastrointest Liver Physiol. 2007; 293(6): G1234-43; Kweon M N, et al. J Clin Invest. 2000; 106(2): 199-206). In patients with food allergies, increased numbers of activated T cells have been correlated with elevated levels of Th2 cytokines as well as the degree of gastrointestinal (GI) inflammation and dysfunction (Eigenmann P A. Pediatr Allergy Immunol 2002; 13:162-71; Eigenmann P A, et al. Adv Exp Med Biol 1996; 409:217). It has been shown that after treatment with oral peanut immunotherapy, levels of peanut-specific Th2-cytokine (IL-4 and IL-5) production by peripheral blood mononuclear cells (PBMCs) was significantly decreased in children with peanut anaphylaxis (Blumchen K, et al. J Allergy Clin Immunol. 2010; 126(1):83-91).
More evidence in humans and mice has shown that Th17 cells, a novel subset of IL-17-producing CD4+T cells, play an important role in the pathogenesis of immune-mediated diseases, including asthma and inflammatory bowel disease (IBD) (Tesmer L A, et al. Immunol Rev. 2008, 223:87-113; Kolls J K and Linden A. Immunity. 2004, 21:467-476). Th17 cells exist and are found constitutively in the small intestine of naive mice housed under conventional conditions (Ivanov I I, et al. Cell. 2006; 126(6): 1121-33). Increased levels of IL-17A (a member of the IL-17 family) have been found in the small intestine of peanut allergy mouse models as well as in the small intestine or in the peripheral blood of food allergy patients (Wang M, et al. J. Allergy Clin Immunol 2010, 126 (2): 306-316). The level of IL-17A is associated with the severity of diarrhea and intestinal inflammation. These data suggested that CD4+T cells that produce Th2 and Th17 cytokines play an important role in food allergy. However, the signal pathway involved in Th2-, Th17-cells responding to allergic food reactions has not been well defined.